ATG proteins mediate efferocytosis and suppress inflammation in mammary involution

Involution is the process of post-lactational mammary gland regression to quiescence and it involves secretory epithelial cell death, stroma remodeling and gland repopulation by adipocytes. Though reportedly accompanying apoptosis, the role of autophagy in involution has not yet been determined. We now report that autophagy-related (ATG) proteins mediate dead cell clearance and suppress inflammation during mammary involution. In vivo, Becn1^+/? and Atg7-deficient mammary epithelial cells (MECs) produced ‘competent’ apoptotic bodies, but were defective phagocytes in association with reduced expression of the MERTK and ITGB5 receptors, thus pointing to defective apoptotic body engulfment. Atg-deficient tissues exhibited higher levels of involution-associated inflammation, which could be indicative of a tumor-modulating microenvironment, and developed ductal ectasia, a manifestation of deregulated post-involution gland remodeling. In vitro, ATG (BECN1 or ATG7) knockdown compromised MEC-mediated apoptotic body clearance in association with decreased RAC1 activation, thus confirming that, in addition to the defective phagocytic processing reported by other studies, ATG protein defects also impair dead cell engulfment.

Using two different mouse models with mammary gland-associated Atg deficiencies, our studies shed light on the essential role of ATG proteins in MEC-mediated efferocytosis during mammary involution and provide novel insights into this important developmental process. This work also raises the possibility that a regulatory feedback loop exists, by which the efficacy of phagocytic cargo processing in turn regulates the rate of engulfment and ultimately determines the kinetics of phagocytosis and dead cell clearance.     

Blockade of 6-phosphogluconate dehydrogenase generates CD8+ effector T cells with enhanced anti-tumor function

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Specific inhibition of hypoxia inducible factor 1 exaggerates cell injury induced by in vitro ischemia through deteriorating cellular redox environment

Hypoxia inducible factor 1 (HIF-1) has been suggested to play a critical role in the fate of cells exposed to hypoxic stress. However, the mechanism of HIF-1-regulated cell survival is still not fully understood in ischemic conditions. Redox status is critical for decisions of cell survival, death and differentiation. We investigated the effects of inhibiting HIF-1 on cellular redox status in SH-SY5Y cells exposed to hypoxia or oxygen and glucose deprivation (OGD), coupled with cell death analyses. Our results demonstrated that inhibiting HIF-1α expression by HIF-1α specific small interfering RNA (siRNA) transfection increased reactive oxygen species generation, and transformed the cells to more oxidizing environments (low GSH/GSSG ratio, low NADPH level) under either hypoxic or OGD exposure. Cell death increased dramatically in the siRNA transfected cells, compared to non-transfected cells after hypoxic/OGD exposures. In contrast, increasing HIF-1α expression by desferrioxamine, a metal chelator and hydroxylase inhibitor, induced a more reducing environment (high GSH/GSSG ratio, high NADPH level) and reduced cell death. Further studies showed that HIF-1 regulated not only glucose transporter-1 expression, but also the key enzymes of the pentose phosphate pathway such as glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. These enzymes are important in maintaining cellular redox homeostasis by generating NADPH, the primary reducing agent in cells. Moreover, catalase significantly decreased cell death in the siRNA-transfected cells induced by hypoxia and OGD. These results suggest that maintenance of cellular redox status by HIF-1 protects cells from hypoxia and ischemia mediated injuries.     

Metabolism addiction in pancreatic cancer

Pancreatic ductal adenocarcinoma, an aggressively invasive, treatment-resistant malignancy and the fourth leading cause of cancer deaths in the United States, is usually detectable only when already inevitably fatal. Despite advances in genetic screening, mapping and molecular characterization, its pathology remains largely elusive. Renewed research interest in longstanding doctrines of tumor metabolism has led to the emergence of aberrant signaling pathways as critical factors modulating central metabolic networks that fuel pancreatic tumors. Such pathways, including those of Ras signaling, glutamine-regulatory enzymes, lipid metabolism and autophagy, are directly affected by genetic mutations and extreme tumor microenvironments that typify pancreatic tumor cells. Elucidation of these metabolic networks can be expected to yield more potent therapies against this deadly disease.     

The enhancement of glucose uptake caused by the collapse of gap junction communication is due to an increase in astrocyte proliferation

We have previously shown that several gap junction uncouplers increase the uptake of glucose in astrocytes. The aim of the present work was to study whether the increase in glucose uptake was a consequence of the inhibition of gap junction communication and the purpose of this effect. Our results show that alpha-glycyrrhetinic acid and endothelin-1 increase the uptake of glucose in highly, but not in poorly, coupled astrocytes. This effect depended on connexin 43 levels and was abolished when the inhibition of gap junction communication was prevented by tolbutamide or ouabain. The inhibition of gap junctions increased the rate of glucose incorporation into DNA and RNA, which was inhibited by treatment with dehydroepiandrosterone, an inhibitor of glucose-6-phosphate dehydrogenase, the regulatory enzyme of the pentose phosphate pathway. The inhibition of gap junctions significantly increased astrocyte proliferation, which was counteracted by tolbutamide. These effects were not observed in poorly coupled astrocytes expressing low levels of connexin 43. The increase in astrocyte proliferation caused by gap junction inhibition was prevented when either glucose uptake or the pentose phosphate pathway were inhibited. We conclude that the inhibition of gap junction communication induces astrocyte proliferation, resulting in an enhancement of glucose uptake and its utilization through the pentose phosphate pathway to provide ribose-5-phosphate for the synthesis of nucleic acids.     

Regulation of the pentose phosphate pathway in cancer

Energy metabolism is significantly reprogrammed in many human cancers, and these alterations confer many advantages to cancer cells, including the promotion of biosynthesis, ATP generation, detoxification and support of rapid proliferation. The pentose phosphate pathway (PPP) is a major pathway for glucose catabolism. The PPP directs glucose flux to its oxidative branch and produces a reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), an essential reductant in anabolic processes. It has become clear that the PPP plays a critical role in regulating cancer cell growth by supplying cells with not only ribose-5-phosphate but also NADPH for detoxification of intracellular reactive oxygen species, reductive biosynthesis and ribose biogenesis. Thus, alteration of the PPP contributes directly to cell proliferation, survival and senescence. Furthermore, recent studies have shown that the PPP is regulated oncogenically and/or metabolically by numerous factors, including tumor suppressors, oncoproteins and intracellular metabolites. Dysregulation of PPP flux dramatically impacts cancer growth and survival. Therefore, a better understanding of how the PPP is reprogrammed and the mechanism underlying the balance between glycolysis and PPP flux in cancer will be valuable in developing therapeutic strategies targeting this pathway.     

A critical role of glucose-6-phosphate dehydrogenase in TAp73-mediated cell proliferation

The pentose phosphate pathway (PPP) provides ribose and NADPH that support biosynthesis and antioxidant defense. Our recent findings suggest that the p53-related protein TAp73 enhances the PPP flux. TAp73 stimulates the expression of glucose-6-phophate dehydrogenase (G6PD), the rate-limiting enzymes of the PPP. Through this regulation, TAp73 promotes the accumulation of macromolecules and increases cellular capability to withstand oxidative stresses. TAp73 also regulates other metabolic enzymes, and the relative importance of these targets in TAp73-mediated cell growth is not well understood. Here we show that, like in other cell lines, TAp73 is required for supporting proliferation and maintaining the expression of G6PD in the human lung cancer H1299 cells. Restoration of G6PD expression almost fully rescues the defects in cell growth caused by TAp73 knockdown, suggesting that G6PD is the major proliferative target of TAp73 in these cells. G6PD expression is elevated in various tumors, correlating with the upregulation of TAp73. These results indicate that TAp73 may function as an oncogene, and that G6PD is likely a focal point of regulation in oncogenic growth.     

Metabolic flux maps comparing the effect of temperature on protein and oil biosynthesis in developing soybean cotyledons

Metabolic flux maps developed from ¹³C metabolic flux analysis (¹³C MFA) are effective tools for assessing the response of biological systems to genetic or environmental perturbations, and for identifying possible metabolic engineering targets. Experimental treatments were designed to distinguish between temperature effects prior to, and during incubation in vitro , on primary metabolism in developing soybeans. Biomass accumulation increased with temperature as did carbon partitioning into lipids. The flux through the plastidic oxidative pentose phosphate pathway (pgl^P) relative to sucrose intake remained fairly constant [∼56% (±24%)] when cotyledons were transferred from an optimum growth temperature to varying temperatures in in vitro culture, signifying a rigid node under these conditions. However, pgl^P flux ranged from 57 to 77% of sucrose intake when growth temperature in planta varied and were cultured in vitro at the same temperature (as the plant), indicating a flexible node for this case. The carbon flux through the anaplerotic reactions catalysed by plastidic malic enzyme (me^P), cytosolic phosphoenolpyruvate (PEP) carboxylase and the malate (Mal) transporter from the cytosol to mitochondrion varied dramatically with temperature and had a direct influence on the carbon partitioning into protein and oil from the plastidic pyruvate (Pyr) pool. These results of the in vitro culture indicate that temperature during early stages of development has a dominant effect on establishing capacity for flux through certain components of central carbon metabolism.     

Evolution of the glucose-6-phosphate isomerase: the plasticity of primary metabolism in photosynthetic eukaryotes

Glucose-6-phosphate isomerase (GPI) has an essential function in both catabolic glycolysis and anabolic gluconeogenesis and is universally distributed among Eukaryotes, Bacteria, and some Archaea. In addition to the cytosolic GPI, land plant chloroplasts harbor a nuclear encoded isoenzyme of cyanobacterial origin that is indispensable for the oxidative pentose phosphate pathway (OPPP) and plastid starch accumulation. We established 12 new GPI sequences from rhodophytes, the glaucophyte Cyanophora paradoxa, a ciliate, and all orders of complex algae with red plastids (haptophytes, diatoms, cryptophytes, and dinoflagellates). Our comprehensive phylogenies do not support previous GPI-based speculations about a eukaryote-to-prokaryote horizontal gene transfer from metazoa to gamma-proteobacteria. The evolution of cytosolic GPI is largely in agreement with small subunit analyses, which indicates that it is a specific marker of the host cell. A distinct subtree comprising alveolates (ciliates, apicomplexa, Perkinsus, and dinoflagellates), stramenopiles (diatoms and Phytophthora [oomycete]), and Plantae (green plants, rhodophytes, and Cyanophora) might suggest a common origin of these superensembles. Finally, in contrast to land plants where the plastid GPI is of cyanobacterial origin, chlorophytes and rhodophytes independently recruited a duplicate of the cytosolic GPI that subsequently acquired a transit peptide for plastid import. A secondary loss of the cytosolic isoenzyme and the plastid localization of the single GPI in chlorophycean green algae is compatible with physiological studies. Our findings reveal the fundamental importance of the plastid OPPP for Plantae and document the plasticity of primary metabolism.     

Functional Genomics Reveals Synthetic Lethality between Phosphogluconate Dehydrogenase and Oxidative Phosphorylation

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Photosynthesis and carbohydrate metabolism in tobacco leaves during an incompatible interaction with Phytophthora nicotianae

The metabolic and cellular changes in source leaves of Nicotiana tabacum L. cv SNN during an incompatible interaction with Phytophthora nicotianae van Breda de Haan were investigated and compared with defence reactions. Hypersensitive cell death was preceded by a rapid and highly localized shift to non-assimilatoric metabolism. During the first 6 h post infection (hpi), reactive oxygen species (ROS) accumulated. Callose was deposited at the interface of adjacent mesophyll cells (≥1 hpi), the export of sucrose collapsed and its content in the apoplast increased. Stomata closed and photosynthetic flux was reallocated from CO₂ assimilation in favour of photorespiration. This was accompanied by an increase in respiration, glucose-6-phosphate dehydrogenase (G6PDH) activity, apoplastic invertase and hexose content. Later (>6 hpi) the photosynthetic electron transport chain was interrupted and photosynthesis completely collapsed. This was accompanied by a further increase in apoplastic invertase and carbohydrates, respiration and oxidative pentose phosphate pathway (OPPP) and followed by further burst in ROS release. Hypersensitive cell death did not appear until photosynthesis completely declined. Photosynthesis was visualized by chlorophyll-a fluorescence imaging on a macro- and microscopic scale. Decline in photosynthesis and defence reactions were highly localized processes, which occur in single mesophyll cells. We propose that in photoautotrophic leaves, photosynthesis and assimilatory metabolism must be switched off to initiate respiration and other processes required for defence. An early blockage of intercellular sugar transportation, due to callose deposition, in conjunction with enhanced apoplastic invertase activity could facilitate this metabolic shift.     

重组菌Ralstonia eutropha W50-EAB D-木糖代谢相关的限速靶点

【目的】通过代谢工程改造真养罗氏菌(Ralstonia eutropha)W50-EAB木糖代谢的相关限速靶点,进一步提高R.eutropha W50-EAB的D-木糖利用效率,为获得高效利用纤维素水解液的菌株奠定基础。【方法】利用PCR技术扩增R.eutropha转酮酶基因tkt A,cbb T2和转醛酶基因tal,将扩增的tkt A,cbb T2和tal基因分别构建到表达载体p BBR1MCS-3上,获得重组质粒p WL1-TKT,p WL1-CBBT2,p WL1-TAL。通过电转的方式将质粒分别转化W50-EAB获得重组菌W50-KAB,W50-CAB和W50-TAB。利用基因敲除的方法,获得醛还原酶基因h16_A3186敲除株W50’-EAB。通过电转的方式将重组质粒p WL1-TAL导入敲除株W50’-EAB获得重组菌株W50’-TAB。通过摇瓶发酵研究重组菌株W50-KAB,W50-CAB,W50-TAB,W50’-EAB以及W50’-TAB的发酵特性。【结果】酶活分析结果表明,转酮酶和转醛酶基因实现表达。摇瓶发酵结果表明,转酮酶基因过表达菌株W50-KAB和W50-CAB相比于对照菌株W50-EAB/p3,表现出降低的木糖利用能力;而转醛酶基因过表达重组菌株W50-TAB以及敲除菌株W50’-EAB对木糖的利用得到一定的提高。在0.1 mol/L木糖的发酵培养基中,W50-EAB的最大比生长速率为0.035 h-1,PHB干重比为16.2±1.01%;而W50-TAB的最大比生长速率提高到0.039 h-1,PHB干重比达到20.5±0.76%;醛还原酶基因敲除菌株W50’-EAB最大比生长速率提高到0.040 h-1,PHB含量提高到19.8±1.05%。结果显示转醛酶基因的过表达与醛还原酶基因的敲除对木糖利用均表现出一定的优势,将这两种优势组合获得菌株W50’-TAB,摇瓶发酵分析结果为最大比生长速率达到0.042 h-1,PHB积累达到27.9±0.47%,相比于对照菌株提高了72.2%。另外,在含有葡萄糖(0.01 mol/L)和木糖(0.09 mol/L)的混合糖培养下,重组菌株W50-TAB,W50’-EAB和W50’-TAB相比于在纯木糖培养下都表现出更高的生物量和胞内PHB积累量。【结论】磷酸戊糖途径关键酶转醛酶基因的过表达加速了木糖代谢流,从而可以高效利用木糖积累一定量的PHB。醛还原酶对木糖代谢有阻碍作用,敲除该酶基因后木糖代谢能力有了一定的提高,而两者协同作用可以进一步提高重组菌株的木糖利用效率和PHB积累能力。     

Amyloid beta peptide (1–42)‐mediated antioxidant imbalance is associated with activation of protein kinase C in red blood cells

Glycolysis and pentose phosphate pathway (PPP) in red blood cell (RBC) are modulated by the cell oxygenation state. This metabolic modulation is connected to variations in intracellular nicotinamide adenine dinucleotide phosphate‐reduced form (NADPH) and adenosine triphosphate (ATP) levels as a function of the oxygenation state of the cell, and, consequently, it should have physiologic relevance. In the present study, we analysed the effects of amyloid beta peptide (1–42) (Abeta) on RBC metabolism and its relationship with the activity of protein kinase C (PKC). Our results showed that metabolic response to Abeta depended on the degree of cell oxygenation. In particular, under high O₂ pressure, in Abeta‐treated RBC, glucose metabolized through PPP approached that metabolized by RBC under low O₂ pressure, differently to that observed in untreated cells. The effect of Abeta on RBC metabolism was paralleled by increase in PKC enzyme activity, but cytosolic Ca2+ concentration does not seem to be involved in this mechanism. Incubation of Abeta‐treated RBC with a specific inhibitor of PKC partially restores PPP flux. A possible rationalization of the different metabolic behaviours shown by RBC following Abeta treatment is proposed. It takes into account the known post‐translational modifications to cytoskeleton proteins induced by PKC. The reduction in PPP flux may lead to a weakened defence system of antioxidant reserve in RBC, becoming a source of reactive species, and, consequently, its typical, structural and functional features are lost. Therefore, oxidative stress may outflow from the RBC and trigger damage events in adjacent cells and tissue, thus contributing to vascular damage. Copyright © 2015 John Wiley & Sons, Ltd.     

溶氧振荡对聚β羟基丁酸混合培养过程的调控作用

研究生物可降解塑料PHB(Poly β-hydroxy butyrate)生产的低成本和高产率发酵。采用廉价碳源——废食品糕点作为原料,其中同时具有葡萄糖和乳酸。培养是在同一5L发酵罐中先后接入2种细菌,先由乳酸杆菌Lactobacillus delbrueckii消耗葡萄糖产生乳酸,再由真养产碱菌Ralstonia eutrophus消耗乳酸产生PHB。本文应用混沌控制理论设计溶氧振荡来协调混生菌矛盾的生理需求,同时改变振荡节律激励细胞的代谢能。考虑真养产碱菌在乳酸里的代谢,有异养和真养两种途径,其中两种重要代谢通量,生物氧化能ATP和还原能NADPH,能够反映混生菌细胞的生理状态,二者都与供氧密切有关。实验通过取样进行细胞破碎后,由荧光分析仪监测不同发酵阶段的ATP和NADPH,发现2种代谢能随着溶氧控制的不同节律变化而起伏,溶氧节律振荡能够使混合培养的PHB的浓度比常规供氧方法提高1倍。     

Carbon metabolism of chloroplasts in the dark: Oxidative pentose phosphate cycle versus glycolytic pathway

The conversion of U-labelled [¹⁴C]glucose-6-phosphate into other products by a soluble fraction of lysed spinach chloroplasts has been studied. It was found that both an oxidative pentose phosphate cycle and a glycolytic reaction sequence occur in this fraction. The formation of bisphosphates and of triose phosphates was ATP-dependent and occurred mainly via a glycolytic reaction sequence including a phosphofructokinase step. The conversion, of glucose-6-phosphate via the oxidative pentose phosphate cycle stopped with the formation of pentose monophosphates. This was found not to be because of a lack in transaldolase (or transketolase) activity, but because of the high concentration ratios of hexose monophosphate/pentose monophosphate used in our experiments for simulating the conditions in whole chloroplasts in the dark. Some regulatory properties of both the oxidative pentose phosphate cycle and of the glycolytic pathway were studied.Abbreviations DHAP dihydroxyacetone phosphate - GAP 3-phosphoglyceraldehyde - PGA 3-phosphoglycerate - HMP hexose monophosphates - including F6P fructose-6-phosphate - G6P glucose-6-phosphate - GIP glucose-1-phosphate - 6-PGL phosphogluconate - PMP pentose monophosphates - including R5P ribose-5-phosphate - Ru5P ribulose-5-phosphate - X5P xylulose-5-phosphate - E4P erythrose-4-phosphate - S7P sedoheptulose-7-phosphate - FBP fructose-1,6-bisphosphate - SBP sedoheptulose-1,7-bisphosphate - RuBP ribulose-1,5-bisphosphate     

p53 Suppresses Metabolic Stress-Induced Ferroptosis in Cancer Cells

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Vanadium accumulation in ascidian coelomic cells is associated with enhanced pentose phosphate pathway capacity but not overall aerobic or anaerobic metabolism

Some suborders of ascidians (sea squirts) accumulate remarkable levels of the heavy metal vanadium while others accumulate negligible amounts. The function of this vanadium is unclear, but enhanced pentose phosphate pathway (PPP) has been implicated in its reduction and accumulation. We compared aspects of intermediary metabolism in coelomic cells from ascidian species that have a wide range of vanadium accumulation including non-accumulators. All species appear to have similar aerobic poise with no apparent link to vanadium accumulation. Similarly, all species examined have a limited anaerobic poise that does not seem to relate to vanadium levels. Based on the activities of phosphoglucose isomerase and glucose-6 phosphate dehydrogenase we demonstrate that, relative to the capacity for entry into glycolysis, vanadium-accumulating species have enhanced capacity to metabolize glucose-6 phosphate via the PPP compared to non-accumulators. This finding provides the first comparative support for enhanced PPP capacity linked to vanadium accumulation in tunicates.     

Functional Electron Microscopy in Studies of Plant response and adaptation to Anaerobic Stress

This article reviews the contribution made by functional electronmicroscopy towards identifying and understanding the reactionsof plant roots and shoots to anaerobic stress. Topics examinedinclude: (1) unexpected hypersensitivity, rather than hyper-resistance,to anoxia of root tips of flooding-tolerant plants; (2) protective,rather than damaging, effects of a stimulated energy metabolism(glycolysis and fermentation) under anaerobic conditions; (3)the concept of two main strategies of plant adaptation to anaerobicenvironments, namely avoidance of anaerobiosis on the wholeplant level, termed ‘apparent’ tolerance, and metabolicadaptation at the cellular and molecular levels, termed ‘true’tolerance; (4) the importance of protein synthesis during hypoxiaand anoxia for enhanced energy production and metabolic adaptation;(5) a general adaptive syndrome in plants to stress at the ultrastructurallevel and a possible molecular mechanism for its realizationunder anoxia; (6) the physiological role of anaerobically synthesizedlipids and nitrate as alternative electron acceptors in an oxygen-freemedium; and (7) the selection of cell lines derived from calluscultures that possess enhanced tolerance to anoxia and can regeneratewhole plants with improved tolerance of soil waterlogging.